The principle of affinity chromatography is based on the phenomenon of molecular recognition. A ligand immobilised on a support matrix is able to form a specific, reversible interaction with a target molecule in the presence of a mixture of other molecules. The nature of the interaction may be hydrogen-bonding, electrostatic forces, stacking as a result of favourable geometry or any other aspect that encourages the host-target relationship. Once bound to the target, the ligand-target interaction should be sufficiently strong to allow the removal of the other contaminant molecules from a mixture while keeping the ligand-target complex intact. However, the binding must be sufficiently weak such that an induced change in local, e.g. buffer conditions causes disruption to the interaction, thus releasing the target molecule in its now purified form. The immobilised ligand, now devoid of protein, can be used again for a subsequent purification. Desirable properties of an affinity ligand include chemical and thermal stability, and high selectivity.
The ligand may be designed to fit a particular target molecule by use of molecular modelling or it may be selected by screeing combinatorial libraries. The combinatorial approach affords a large number of ligands which may be constructed to incorporate a variety of chemical moieties including hydrophilic, hydrophobic, charged or neutral groups or a mixture thereof. Combinatorial libraries may conveniently be synthesised by incorporating commercially available compounds, including amino acids, carboxylic acids and amines, in a step-wise synthesis directly on the surface of the support matrix. Alternatively, the ligand may be synthesised and subsequently attached to a support matrix.
Cyanuric chloride, or 2,4,6-trichlorotriazine (hereinafter referred to as triazine), is a symmetrical molecule. 2,4,6-Trisubstituted triazines can readily be generated by reaction of cyanuric chloride with nucleophiles such as amine compounds. Triazine derivatives are useful as adsorbents and for other purposes; see WO-A-97/10887 and WO-A-00/67900.
Affinity ligand libraries may be built up on a hydroxylic support such as cross-linked agarose. To date, all triazine-based ligand libraries built up on agarose have comprised a support with a single substituted triazine component (see WO-A-97/10887) or with macrocyclic rings incorporating triazine groups (see WO-A-01/42228).